2.3. Animal models for transient focal cerebral ischaemia

Transient middle cerebral artery occlusion (MCAO) was induced in both ARRB2^−/− and WT mice (25‐28 g) as described previously.5 All surgeries were performed under sodium pentobarbital anaesthesia and all efforts were made to minimize suffering. After 2 hours of MCAO, reperfusion was initiated by the thread careful withdrawal. As an extrinsic ligand of NOD2, muramyl dipeptide (MDP, Sigma‐Aldrich, A9519) was intraventricularly administered to mice 30 minutes before MCAO. MDP (200 μmol/L in normal saline [NS]) or the vehicle (NS) was injected using a Hamilton syringe (3 μL, 0.4 mm posterior and 1.0 mm lateral to the bregma and 2.2 mm from the duramater). After reperfusion, the mice were anaesthetized, perfused with NS and then decapitated. The brains were immediately frozen in liquid nitrogen for the following studies.